Z . C O L IUsing frying oil to produce trans-zeatin in

an engineered strain of E. coli

Colorado State University iGEM

Giant Jamboree 2015

Background

3 billion gallons produced per year in US[1]

Breakdown used frying oil to produce zeatin

1. US EPA. “Learn About Biodiesel”. Web. 8 Jan 2014. Retrieved 20 Aug 2014.

2. Photograph courtesy of Dan Dickinson. Used with permission under creative commons with attribution.

Our Project

ExtracellularBreakdown

TransportMEP

PathwayZeatin

IntracellularBreakdown

Why frying oil?

High energy

CheapFatty acids { } Glycerol

Limiting Steps in Breakdown

Breakdown of Frying Oil Components

Breakdown of Fatty Acids

Transport of Fatty Acids

Breakdown of DAGs

Lipase from Bacillus stearothermophilus

Turns DAGs into FAs and Glycerol

http://www.emhmagazine.com/uploaded/images/123035292.jpg

Transport of LCFAs into Cell

Natural Transporter: long-chain fatty acid outer membrane porin

Encoded for by fadL

Increased Breakdown of FAs

Beta-oxidation pathway

fadD encodes limiting enzyme

Fatty acyl-CoA synthetase

faddfadD Experiment

Growth Experiment with Used Frying Oil

Growth Experiment with Used Frying Oil

Growth Experiment with Used Frying Oil

Initial 50% Solution 50% Solution after 12 hours of Growth

Future Improvements

Quantitative vs. Qualitative Data

Cell Growth Measurement

Optimize Feed and Growth Conditions

Metabolic Engineering

Trans-zeatin

Very expensive

Currently only used in research

Many other applications

https://en.wikipedia.org/wiki/Zeatin

Plant Growth Experiment

Biological Activity of the tzs Gene of Nopaline Agrobacterium tumefaciens GV3101 in Plant Regeneration and Genetic Transformation (Zhao-Fen Han et al., 2013)

MEP Pathway

D-Glyceraldehyde 3-phosphate Pyruvate

MEP

HMBDP

http://www.pet.hw.ac.uk/research/fast1/research/res_images/chem_inhibitors/hmdp.gif

Biosynthesis PathwayZeatin Biosynthesis

Trans-zeatin Synthase

LOGzeatin

https://en.wikipedia.org/wiki/Zeatin

zeatin riboside 5’-phosphate N6-dimethylallyladeninehydroxylation

HMBDPhttp://www.pet.hw.ac.uk/research/fast1/research/res_images/chem_inhibitors/hmdp.gif

Characterization

Zeatin Construct Growth Experiment

Extraction methods Solid Phase Extraction Methods

1. Column Wash

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z

z

z

z

z

z

2. Bind Supernatant 3. Wash

zz

z

z

z

z

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4. Elute

zz z

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Zeatin HPLC Results

Design Considerations

http://instalacionesgrau.com/en/products/bioreactores/

https://pbs.twimg.com/media/CE4Ad2JWAAA6Rp4.jpg

KillerRed

Killer Red Literature

Bulina, M., et. al. "A genetically encoded photosensitizer.” Nature Biotechnology, 95-99. 2005. October 2014.

Kill Switch

• Functional improvement• Removed trp promoter

• Characterization improvement• Collected data on efficacy

KillerRed Improvement

Control KillerRed

Kill Switch Constructs

Improved characterization of KillerRed biobrick

Suspected mutation

Working in a jury-rigged darkroom with KillerRed

interlabInterlab Study

Research

Industry

Public

Spoke with experts and compiled articles on:

Laws and regulation of GMOs

Commercialization and industry

Intellectual property

Bridging the Gap

EntrepreneuershipEntrepreneurship

• Construction and submission of multiple BioBrick Parts (Bba_KI702000-Bba_K1702005)

• Experimental validation of fatty acid breakdown (BBa_K1702001, Bba_K1702002)

• Addressed questions beyond the bench about IP, commercialization, and bridging the gap between academia, industry, and the public

• Used beyond the bench IP research to file provisional patent

• Improved function and characterization of previously existing BioBrick Part, KillerRed (Bba_K1495000)

• Functional prototype of E. coli growing on frying oil

Key Achievements

Key challenges

• Reworking breakdown construct ideas after fadDexperiment

• Inconclusive results about trans-zeatin production

• Proper handling and experimental design for testing KillerRed

Key Challenges

Thank You

Dr. Christie Peebles, Dr. Tom Santangelo, and Dr. Ashok Prasad

Fatty acid growth experimentsLac Promoter

"Lac operon-2010-21-01" by Lac_operon.png: G3proderivative work: Tereseik (talk) - Lac_operon.png. Licensed under CC BY 2.0 via Commons - https://commons.wikimedia.org/wiki/File:Lac_operon-2010-21-01.png#/media/File:Lac_operon-2010-21-01.png

Growth Experiment with 100% Used Frying Oil

Before Inoculation

After 4 hours of growth

Fatty acid growth experimentsFatty Acid Growth Experiment

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0

0.005

0.01

0.015

0.02

0.025

0.03

0 2 4 6 8 10 12 14 16 18 20

OD

60

0

Time [hr]

E. coli Growth on 0.04% Used Frying oil

fadD + fadL

fadD

fadL

lac Promoter

Why Zeatin?

Benefits:• Increase lateral bud growth• Increases immunity against pathogenic attack on plants[1]

• Anti-aging properties• Green (natural) product• Synthetic production is complex and low yield

Competitors:Benzyl adenine• Synthetic• Not as well researched• Zeatin is 10x more effective[2]

1. Grubkinsky, Dominik et al. ‘Cis- and trans- zeatin differentially modulate plant immunity’. Plant Signal Behav. 2013 8(7); e247982. Bajaj, Y.P.S. Plant Protoplasts and Genetic Engineering IV 2012. Springer Science and Business Media.

Zeatin Biosynthesis

MEP Pathway fullMEP Full Pathway

Tz bioreactor with dataTrans-zeatin Bioreactor Data

Killer red dataKiller Red Data